ABSTRACT The pathogenic amebae, Entamoeba histolytica and Naegleria fowleri are deadly human pathogens with limited therapeutic options. Entamoeba causes dysentery and liver abscesses in 50 million people annually. Naegleria, causes primary amebic meningoencephalitis (PAM), a rare but rapidly fatal disease. Despite being important human pathogens, these organisms have very limited therapeutic options, and no significant drug development pipeline exists. For Naegleria there is no drug regimen that reliably cures disease. Only a handful of patients have survived; >97% fatality rate is the current standard. For Entamoeba there is a need for improved drugs that overcome side effects, problems with treating pregnant and lactating women, concern for emerging drug resistance, and a complex treatment regimen for the two life cycle stages. Thus, there is a critical need for new drugs against these pathogens. We determined that fumagillin and its derivatives, which target MetAP2 in other systems, have good in vitro activity against Entamoeba and Naegleria. The in vitro activity against Naegleria is exceptional and given the current abysmal treatment options, there is great motivation to identify and optimize compounds for in vivo efficacy in an animal model of Naegleria infection. Our goal is to validate MetAP2 as the target of fumagillin and its analogs in Entamoeba and Naegleria. Having a demonstrated target will serve as a foundation for further development of MetAP2 inhibitors, as anti-amebic drugs. Our workflow is: Aim 1. Demonstrate that parasite MetAP2 is a viable drug target for Entamoeba and Naegleria, by using biochemical assays and genetic approaches. Aim 2: Screen a larger series of MetAP2 inhibitors for activity against Entamoeba and Naegleria; focusing on drug development for Naegleria, we will identify select compounds with optimal efficacy and appropriate PK with CNS penetration for future efficacy testing in an animal model of Naegleria. Based on our strong preliminary data, experience with ameba biology, access to an extensive MetAP2 compound series, and significant drug development expertise at Stanford and UCSD we are confident of a successful outcome. The R21 mechanism, developed to encourage exploratory research by providing support for early stages of project development, is ideal for this proposal. If a compound lead that is highly effective against Naegleria can be identified, it could lead to a fundamental breakthrough in management of this highly fatal disease. Drugs against Naegleria would be eligible for a fast track drug development including orphan drug and rare pediatric disease designations and compassionate use by the FDA under an expanded access program.